Ultrasound and x-ray imageable poloxamer-based hydrogel for loco-regional therapy delivery in the liver

Intratumoral injections have the potential for enhanced cancer treatment efficacy while reducing costs and systemic exposure. However, intratumoral drug injections can result in substantial off-target leakage and are invisible under standard imaging modalities like ultrasound (US) and x-ray. A thermosensitive poloxamer-based gel for drug delivery was developed that is visible using x-ray imaging (computed tomography (CT), cone beam CT, fluoroscopy), as well as using US by means of integrating perfluorobutane-filled microbubbles (MBs). MBs content was optimized using tissue mimicking phantoms and ex vivo bovine livers. Gel formulations less than 1% MBs provided gel depositions that were clearly identifiable on US and distinguishable from tissue background and with minimal acoustic artifacts. The cross-sectional areas of gel depositions obtained with US and CT imaging were similar in studies using ex vivo bovine liver and postmortem in situ swine liver. The gel formulation enhanced multimodal image-guided navigation, enabling fusion of ultrasound and x-ray/CT imaging, which may enhance targeting, definition of spatial delivery, and overlap of tumor and gel. Although speculative, such a paradigm for intratumoral drug delivery might streamline clinical workflows, reduce radiation exposure by reliance on US, and boost the precision and accuracy of drug delivery targeting during procedures. Imageable gels may also provide enhanced temporal and spatial control of intratumoral conformal drug delivery.

Additional information pertaining to rheological studies can be found in Table S1 and S2).

US imaging assessment with tissue mimicking phantoms
Percent MBs in POL gel was optimized using tissue mimicking phantoms (TMP) (Fig. S10).Imaging 1% MBs produced images with acoustic intensities similar to that for 5%, and 10% MBs in POL gel and were clearly distinguished from background.In addition, the acoustic heterogeneities and entropies were similar to 5%, and 10% MBs in POL.Acoustic heterogeneity measured how much the pixel intensities deviated from the mean pixel intensity in the selected region of interest (ROI).Entropy is the measure of randomness in the position of pixel values in an ROI (Fig. S10).POL with 1% MBs also provided areas with decreased acoustic artifacts such as reflective gray areas deep to the POL location (comet tails).Due to these results, POL gel with % MBs below 1% was further tested ex vivo.

Assessment of US imageability of POL in ex vivo tissue
POL gels with 0.001, 0.01, and 0.1% MBs, were imaged after injection in ex vivo bovine liver.This range was selected for study as these lower % MBs provided a clear delineation of gel from surrounding tissue on US without introducing significant acoustic artifacts (Fig. S12).The entropy of the images of the POL gels, a statistical measure that can be used to characterize the texture of the input image, was lower in POL with 0.01% MBs compared to 0.001% MBs (p = 0.0389) (Fig. S12F).From this optimization, 0.01% MBs formulation was selected for further testing (Fig. S12).
Distance from the POL gel deposition center to the needle tip also remained tended to increase with injection volume (Fig. 4F)., with SEHN distances from 0.3 ± 0.1 to 0.5 ± 0.2 cm, MSHN from 0.9 ± 0.4 to 1.0 ± 0.3 cm, and MPIN-1 cm from 1.0 ± 0.4 to 1.2 ± 0.5 cm for 1 to 4 mL injections, respectively.The relative effects in the distance of the center of the POL injection to the needle tip in the volume suggested to be different depending on the needle device used.The weighted effect of SEHN in the distance to needle tip suggested to be higher compared to the other needle devices (See regression coefficients in Table S3), nevertheless, no differences were obtained (p > 0.3) (Table S4).In addition, the distance of POL center to the needle tip values per mL injected correlated across all the needle devices (Fig. S13).Additional information of statistical analysis pertaining to the distance of needle tip to POL injection center can be found in Table S5 and S6.
The solidities (concavity degree) for the areas analyzed per mL of POL gel are available in Fig. S14.
For the second injection technique, (Fig. 5F), while monitoring the POL deposition, the US image was variably hypoechoic (dark regions) and hyperechoic.The track of injected POL was visible after the needle was removed www.nature.com/scientificreports/due to elimination of needle artifacts.The POL deposition was in form of string of speckles with a 0.7 ± 0.1 cm major axis length and 0.5 ± 0.0 minor axis length.The acoustic heterogeneity was calculated and was 48.8 ± 7.4 a.u and 45.2 ± 6.1 a.u.for the first and second technique, respectively (Fig. 5G).The entropies for both techniques were 7.3 ± 0.1 a.u., and 7.5 ± 0.2 a.u.for the first and second technique respectively (Fig. 5G).

Assessment of US imageability of POL gel containing DOX in ex vivo tissue
POL gel loaded with DOX 10 mg/mL was imageable under US (Fig. 7A) and was visible over 100 min (Fig. 7).
The area of POL based on US imaging did not differ between 0 and 100 min after injection (p > 0.9999) (Fig. 7).

Assessment of US and x-ray imageability of POL gel in ex vivo tissue
POL gel was imageable under US and x-ray-based imaging, both CT and fluoroscopy.Both US and CT imaging showed similar morphology (Fig. 8A and B) and 2D measurements (Fig. 8C-E).Eccentricities were 0.8 ± 0.1, and 0.6 ± 0.2 for 1 mL POL measured from US and CT, respectively (Fig. 8E).For the 3 mL POL injections, the eccentricity values were 0.8 ± 0.1 a.u., and 0.5 ± 0.1 a.u.for US and CT, respectively.The solidities were 0.8 ± 0.0 a.u., and 0.9 ± 0.1 a.u.for 1 mL of POL injected, based on US and CT imaging, respectively (Fig. 8E).Finally, the solidities for 3 mL of POL injected were 0.8 + 0.0 a.u. and 0.9 ± 0.0 a.u.based on US and CT imaging, respectively (Fig. 8E).US and CT images of POL co-registered (dual imaging fusion overlapping) injected into ex vivo liver demonstrates dual imageability of POL (Fig. 9A-C).Qualitatively, both co-registered images matched the localization of POL.Linear structures, presumed to be vessels or fascial planes filled with air, were visualized under US imaging, but were not readily visible under CT.
3D distribution of POL from US sweeps with C5-1 transducer were similar to the shape derived from CT. Figure 9D depicts a representative 3D distribution of POL with 0.01% MBs injected with SEHN.The 3D images from US provided irregular and rugose surfaces compared to the smoother surfaces from CT. Volume, sphericity, solidity, and surface-area-to-volume (SA/V) of POL gel with and without DOX revealed slight differences when comparing with US and CT 3D distributions (Table 2, I Fig. 10).Overall, the volumes of POL injections obtained with CT imaging (Fig. 10A and B), were lower than those of US imaging.
The sphericities of POL obtained from CT imaging were generally higher compared to US imaging (Fig. 10C  and D).In addition, solidities of POL 3D distributions were higher from CT imaging compared to US imaging (Fig. 10E and F), but that could perhaps be due to volume averaging and lack of detection of small vessel incursion on CT.Finally, the SA/V tended to be higher for 3D distribution of POL injections obtained with US imaging compared to CT (Fig. 10G and H).However, the differences between results with US and CT could be due to the irregularity of the rendered surface on US.Many factors might contribute to the smoothness of the edge, including surface reconstructions, volume averaging, and smoothing algorithms.
There were some differences on volume, sphericity, solidity, and SA/V parameters in the majority of the formulations of POL with or without DOX after injection with SEHN or MSHN (Fig. 10).

Discussion
The purpose of this study was to develop, optimize.and characterize gels that are imageable under US and x-ray/ CT, utilizing POL as a potential drug carrier, such as for anti-cancer treatments.POL provided controlled drug release in vitro, even when mixed with the US, MBs, and iodixanol.The gel injectability was tested across various   needle devices, showing that its distribution, both in 3D and 2D formats, were predictable and visible using US and CT in ex vivo and postmortem animal models.Liao et al. 39 conducted a similar study with varying POL concentrations from 2 to 17% and incorporated MBs in the gel.The authors reported a slight decrease of gelation temperature value compared to formulations without MBs.However, our gel and data showed the gelation temperature tended to increase with increased MB content.The stability of MBs was evidenced by optical micrographs of gels containing 1% and 10% of MBs at 37 °C.The addition of iodixanol, MBs, and DOX did not compromise the rheological properties of POL, which remained injectable due to its thixotropic nature (recovery of viscosity post stress), and quick transition from liquid to solid upon the cessation of shear stress.This property is crucial for ensuring that POL remains localized at the physiological temperature of 37°C 30 , maintaining its effectiveness for targeted therapy delivery across a variety of syringe sizes with associated differences in pressure and shear stress.Prior work showed that POL resulted in 1.5-fold lower release rates of DOX over the initial 7 h under identical in vitro conditions compared to another formulation without POL 30 .This finding highlights the advantages of using a gel-based drug delivery system over a conventional percutaneous injection of a free drug.
TMP US studies showed that gels with 1 to 10% MB concentrations were visible, though visibility at 10% MB might be compromised due to higher concentration of MBs reflecting back the majority of the sound waves leading to acoustic shadowing in the far field.These studies helped assess the gel texture and acoustic properties.However, MBs concentrations over 1% led to acoustic artifacts like comet tails and gas shadowing, affecting visibility in the far field 46 .Therefore, to minimize such artifacts, MB concentration in gels was maintained below 1% for optimal imaging in ex vivo liver tissue.Although not designed for vessel embolization, the POL formulations might be used as an imageable embolic agent to occlude vessels.Full exploration of this potential application was beyond the scope of the work.Adjusting POL's MB concentration from 0.001 to 0.1% resulted showing high localization and clarity in ex vivo liver without acoustic artifacts.The choice of % MBs was crucial to balance the acoustic signal for optimal contrast and minimal artifacts, while enhancing texture detail within the gel for better tissue structure and gel-tissue interface visualization.POL with 0.001% MBs showed greater detail and therefore higher entropy than with 0.01% MBs, suggesting a more detailed visualization of tissue structures within the gel.Entropy was used as a simplified method to characterize texture differences at the region of interest (POL deposited in tissue).However, clinical imaging of MBs employs advanced techniques such as power modulation and phase inversion to enhance the visualization of US contrast agents 47 .Contrast-Pulse-Sequence processes the reflections of US pulses to construct images of individual MBs, offering optimized contrast-to-tissue ratio 47 .These commercially available techniques for MBs-contrast-only provide enhanced details on MBs distribution in tissue, thereby improving imaging sensitivity and detection of tissue abnormalities, including those resulting tissue disruption after gel administration.Detailed Techniques for characterizing tissue disruption following gel administration were beyond the scope of this study and require further study.
The selection of needle delivery devices and techniques affected the visibility of POL during image-guided injections.POL injections, especially with MPIN needles, sometimes lost US visibility.This may be due to the high pressure and shear stresses exerted on the MBs during passage through the narrow gage needles, which leads their destruction, suggesting an increase in MBs concentration might be necessary for gel use with the MPIN.Another less critical factor might have been the multi-planar volumetric nature of MPIN injections, that may be more challenging to capture within one ultrasound volume.Nevertheless, in general, POL injections with 0.01% MBs showed consistent results across different needles, with gel dimensions and delivered volumes showing a linear relationship.This predictability, crucial in clinical settings, allows for precise definition of treatment areas under US, enhancing the translatability potential 48 .
For POL injections using MPIN, specific injection protocols of needle retractions and rotations may be needed to optimize reproducibility and desired therapeutic effect 49,50 .Despite reduced visibility under US, POL's deposition could still be monitored in real-time US without ionizing radiation.Generally, gel volumes under 1 mL are less visible than those above 1 mL.
As the gel is injected, lower volumes initially create a cavity within the liver parenchyma, via displacement of tissue.Subsequently, as the volume of the gel increases within the tissue, the gel exerts pressure outward from the cavity, filling the tissue pores.The gel remained localized during this injection process in normal liver.How this might extend to neoplastic tissue adjacent to normal liver remains undefined, but might be influenced by interstitial pressures, diffusion, interface of neoplasm with liver, and the underlying health of adjacent normal liver, e.g., cirrhosis or fatty infiltration.
Percutaneous gel injections can use radial or sequential intratumoral techniques 6,51,52 .For sequential injections, visualizing multiple injections simultaneously under US without interference is crucial.Five transducers studied showed that US visibility of adjacent POL depositions, ex vivo, varies with the beam array used.Curvilinear and microconvex arrays offer broader visualization but less superficial near field detail, whereas linear arrays provide detailed near-field views at the expense of a panoramic and far field penetration and perspective.Choosing the right transducer and optimizing imaging and acoustic parameters like frequency and gain are vital for high-quality images, with the choice also depending on the targets.Having an optimized setup of acoustic parameters and transducer may improve the visibility of any echo changes after gel injections.For this study, depending on the transducer, it was possible to distinguish changes in echotexture and tissue, potentially by formation of cavities filled with gel surrounded by tissue or pores or low-pressure channels filled with gel.
POL with DOX maintained US visibility, acoustic properties, and measured volume, showing stable ex vivo tissue visibility over 1.5 h.This stability may have value in applications requiring procedural monitoring of gel and drug dispersion, such as during multiple needle and catheter local procedures.However, actual visibility will likely vary under perfused in vivo conditions.
POL was successfully visualized using both US and x-ray/CT, showing consistent area, eccentricities, solidities, and dimensions in ex vivo and postmortem animal models for 1 mL and 3 mL injections.This demonstrated the potential for multimodal imaging in guiding intratumoral POL injections, akin to fusion guidance and combination multi-modality practices in biopsies and tumor ablations 40,41 .MBs and iodine as contrast agents were simultaneously incorporated into a hydrogel for potential use in multi-modality image-guided drug delivery.
There are some reports of contrast agents, such as for CT, US, magnetic resonance imaging, fluorescence, or single photon emission computed tomography, being incorporated in gels and tested in animal models for cancer therapy or tissue engineering 53,54 .In addition, MBs have been incorporated in POL for intratympanic applications 39 .Even with this plethora of formulations incorporating multimodal imaging gels, there is still a gap in gel formulation characterization using different needle devices, clinically relevant volumes, and commercial clinical instruments; as well as the incorporation of both MBs and iodine in the same gel.
In clinical practice, needle-based interventions may use US and CT alone or in combination for image guidance, depending on the needs for management of each patient as well as the preferences of the physician.The two modalities are based on distinctly different imaging physics and may provide different information, i.e., a lesion may be seen on one and not the other, as well as differential visualization of areas to be avoided during needle placement.The combination of the POL with MBs and iodixanol permits imaging with both modalities and facile switching between the two as needed for specific workflow needs at specific timepoints in patient care related to injectable therapeutics.Thus, the real time nature of US imaging can be used in conjunction with the intra-procedural CT scans as well with fusion imaging of both US and CT and may impact safe and accurate dosage and planned treatment volume target tissue localization, and perhaps clinical outcomes [55][56][57][58][59][60] .
The 3D morphology of US images for POL containing DOX was similar to that seen with CT imaging, suggesting US could be used to estimate and monitor drug delivery or treatment zones without ionizing radiation, offering a potentially cost-effective option in settings lacking CT or CBCT imaging.However, the volumes www.nature.com/scientificreports/calculated from US 2D sweep were higher than that of CT, therefore, caution must be exercised given the slight disparity.The investigation did not explore the potential for oscillation or cavitation of MBs to trigger drug release bursts, immunomodulation, inflammation or tissue destruction.The key factor for inducing cavitation with medical imaging probes is the Mechanical Index (MI) and its setting level.In this study, the MI was maintained low (1.2-1.4), and even at the highest setting, it would not induce inertial cavitation of the bubbles due to their presence in a viscous gel rather than in blood or aqueous solution 61,62 .
There is a potential for prolonged US imaging to induce minimal stable cavitation of the bubbles.However, distinguishing the effects of this in the release studies would be challenging compared to the natural release rate caused by micellar hydrolysis of the gel.In clinical US imaging scenarios involving this gel (with brief exposure), any effect from stable cavitation would likely be negligible compared to the natural release mechanisms of the gel itself.
The use of optical imaging to characterize DOX distribution in bovine liver tissue provided insights into the DOX distribution at various regions both at and distanced from the injection site.Additionally, optical imaging detected DOX intensity in intravasated material.Delgado et al. employed a similar method to characterize DOX in bovine liver tissue following injection of POL with DOX using SEHN, MSHN, and MPIN needle devices, yielding comparable profiles to those reported in this study 30 .In the present research, we expanded upon this information by examining DOX distribution within the injection site, including leaked material.
There were several limitations to this study.The study relied on in vitro, ex vivo, and post-mortem liver models that lack blood flow or tissue perfusion, which would influence the behavior of the POL delivery and subsequent drug release.However, the aim with several of these studies, i.e., effects of MB and iodixanol on drug release, were intended to establish the effects of the constituents on drug release rather than precise prediction of in vivo behavior.The differences in acoustic properties between liver and tumor tissues could not be addressed, nor were other factors such as blood flow, lymphatic flow, interstitial pressures, fascial planes, diffusion, convection, tissue heterogeneities or viscosity.As further considerations, the properties of the tumor and liver tissue may affect drug release and efflux, perhaps due to heterogeneous tissue properties, tumor fibrosis, liver cirrhosis, drug adsorption by tissue, or elevated interstitial pressure, either intrinsic to the tumor or induced by the gel injection.Thus, further testing in large animal models and appropriate tumor models is a requisite step in assessing appropriateness of this vector for clinical use such as for intratumoral hepatic injections.Such studies would assess POL delivery and drug elution with pharmacokinetic evaluations, providing better representation of clinical drug delivery performance.

Conclusion
In this study, a gel based on POL visible under US and x-ray/CT was developed, optimized, and characterized in vitro,in ex vivo bovine liver, and in postmortem swine liver.The gel demonstrated sustained DOX release in vitro, unaffected by the presence of MBs, and was injectable through a variety of commercial needles due to its ability to recover viscosity after stress.Both US and x-ray/CT imageability were verified in ex vivo and postmortem analyses, and the gel was compatible with electromagnetic navigation for multimodal fusion imaging co-registration and co-display.The potentially predictable distribution of POL in ex-vivo and postmortem tissue enhances its translational potential for clinical use.Additionally, 3D US imaging allowed for monitoring and prediction of injected treatment volumes with fusion guidance without use of ionizing radiation.This research introduces a promising potential tool for image-guided intratumoral injections of anti-cancer agents.
Microbubble preparation: Microbubbles (MBs) were prepared as previously described by Owen et al. 63 Briefly, 0.6 mL of DSPC (25 mg/mL) in chloroform and 0.4 mL of PEG-40-Stearate (10 mg/mL) were mixed in an 8 mL glass vial (Thomas Scientific, Swedesboro, NJ, USA).The organic solvent was evaporated under stirring overnight.The next day, the lipid film was dissolved in a solution consisting of 8:1:1 normal saline, glycerol, and polyethylene glycol, heated to 79 °C, and subsequently ultrasonicated (20 kHz for 2 min, 20% power) with a sonic probe (Q55 Qsonica Sonicators, Newtown, CT, USA) deep in the solution.After sonicating and collapsing the lipid structures, the probe tip was moved to the gas/liquid interface under perfluorobutane bubbling and, the solution was ultrasonicated (20 kHz for 20 s, 90% power).This produced a white solution of MBs, which was then rapidly cooled in ice water.
Gel preparation: As previously reported, POL formulation was prepared using the cold method. 28Briefly, POL powder was mixed with normal saline, and iodixanol (Visipaque 320) was added to achieve a concentration of 22% (w/v) POL and 40 mg/mL iodine in the formulation (POL).The mixture was then stirred at 4 °C for at least 12 h.To prepare POL formulation containing DOX, the above procedure was followed, and doxorubicin was subsequently added to achieve a final concentration of 10 mg/mL, and stirred for 5 days at 4 °C.
Gel preparation with MBs: MBs at a concentration of 1 × 10 9 bubbles/mL 64 were prepared in a saline:glycerol:polyethylene glycol mixture.The mixture was added via a pipette to the POL gel to produce concentrations ranging from 0.001 to 10% (v/v), where the volumetric ratio is the volume of the MBs mixture gel.After depositing the MBs, the solution was gently stirred to thoroughly mix the MBs with the POL gel.Due to the high viscosity and rapid gelation of POL gel, careful stirring was necessary to ensure even distribution of the MBs.For POL with DOX, the MBs were added to the gel product at 4 °C and the liquid material was shaken manually until the MBs were well dispersed.In most cases, MBs were used immediately after preparation, typically within one hour.Before each formulation was loaded into the syringe, the MBs were gently agitated manually and then mixed with POL.
Optical microscopy: MBs, POL, and POL with MBs were visualized using a fluorescence microscope (Imager M2, Zeiss, White Plains, NY, USA) with a 5X objective.MBs were examined using a hemocytometer and counted in MATLAB software (R2020a version, Mathworks, Natick, MA) using a method previously outlined. 64For the POL with MBs, 100 µl was added onto a microscope slide at room temperature.This was then observed under the optical microscope in one area.Subsequently, the slide with the bubble-gel was heated to 37 °C for 10 min, and the same area of the bubble-gel was observed again to determine if the MBs remained present after the temperature transition process from liquid to gel.
Gelation time of POL with MBs: Gelation times were estimated by the inverted tube method. 45Briefly, 400 µl of bubble-gel containing 0, 1, 3, 7, 9, and 10% of MBs (v/v) were added to borosilicate vials.Each vial was equilibrated at room temperature for 5 min and subsequently submerged in a 37 °C water bath.The vials were then inverted while still under water.The time at which the bubble-gel stopped flowing and remained at the bottom of the vial was recorded.
Oscillatory rheology analysis: The determination of the gelation temperatures for both imageable and nonimageable POL variants was conducted using a Discovery HR20 rheometer (TA Instruments, New Castle, DE) equipped with a 25.0 mm stainless steel parallel plate setup maintaining a 500 µm gap.Prior to conducting oscillatory rheology tests, both the parallel plate assembly and the stage holding the samples were coated with mineral oil (conforming to ASTM oil standards).
Determination of gelation temperature: The influence of increased MBs concentration (0, 0.01, 0.01, and 1%, v/v) on the gelation temperature of POL variants devoid of iodixanol, those incorporating iodixanol (40 mg/mL of iodine), and those containing DOX (10 mg/mL) was assessed following the methodology outlined by Baloglu, et al. 65 This involved gradually heating the samples from 5 to 37 °C.During this process, the samples underwent a strain of 0.2% and an angular frequency of 6.0 rad/s.The gelation temperature was defined, in accordance with established literature convention 66,67 , as the midpoint of the storage modulus value (G') from the temperaturedependent rheological data.
Viscoelastic properties: POL containing MBs concentration (0, 0.01, 0.01, and 1%, v/v), iodixanol (40 mg/mL of iodine), and DOX (10 mg/mL) was characterized for viscoelastic and thixotropic properties.A time sweep was performed for 1 h at 0.2% strain and 6 rad/s at 37 °C with a 1000% strain event applied for 1 min followed by a 60 min at 0.2% strain to evaluate G' and G" values.The ƞ* was calculated as: ƞ* was noted right before, during, right after strain, 10 min after strain, and 56 min after strain.Percentage of ƞ* recovered was obtained from the G' value during strain and G' value right after strain.To determine the linear viscoelastic regions, the flow points of POL were determined with amplitude sweeps done from 0.1 to 1000% oscillation strain at 6 rad/s.Flow points were noted when G' = G", tan δ = 1.n = 3 for each measurement.
In vitro drug release: The in vitro drug release of POL with iodixanol (40 mg/mL), MBs concentration (0,0.01,0.01, and 1%, v/v), and DOX (10 mg/mL) was evaluated with a dialysis cassette with a 3.5k molecular weight cut-off (Pur-A-Lyzer Midi 3500, Sigma Aldrich).Using infinite sink conditions, 800 µl of POL with DOX 10 mg/mL was added to the dialysis cassette and incubated at 37 °C for 10 min.The samples were then added to 50 mL conical tubes containing 30 mL of 37 °C normal saline and placed in a shaker (Roto-Therm Plus, Ward's Science, Rochester, NY, USA) (rocking mode,10) maintained at 37 °C.200 µl aliquots were taken at 0, 1, 2, 3, 4, 5, 6, 7, 21, 45, and 69 h with normal saline volume replacement.DOX concentration in each aliquot from POL was calculated from absorbance measurements with a microplate reader (Biotek Cytation 5, Agilent) at λ = 483 nm and by comparison to a calibration series of DOX solutions of known concentration.
Tissue mimicking phantoms (TMP) US imaging: A material reservoir measuring 4.5 × 11 × 4.5 cm was 3D printed (Ultimaker S3, New York, NY, USA) using tough black polylactic acid (PLA) (Ultimaker material, New York, NY, USA).The tissue mimicking phantoms were prepared with agarose 2%.Briefly, 0.5 g of agarose was added to 500 mL of deionized water and solubilized under microwave heating.The solubilized transparent liquid was transferred to the reservoir.Subsequently, 1 × 1 × 2 cm plastic cuvettes were inserted into the liquid agar to form cavities into which the POL could be placed.The solidified agarose was allowed to cool for 1 h at 4 °C.To assess the US B-mode imaging of the MBs and POL with MBs, the mean acoustic intensity was characterized by using a IU22 US system (Philips, Cambridge, MA, USA) with a C5-2 transducer for POL as a liquid (T = 21 °C) and POL as a gel (T = 37 °C), The transducer was positioned perpendicularly to the TMP and the imaging parameters were frequency = 77 Hz, 40%, and C = 54.
In addition to acoustic intensity, the acoustic heterogeneity of agarose-based TMP US images was obtained by determining the standard deviation of the gray-scale histogram pixel intensity of a rectangular region of interest (ROI) with 1 cm 2 area.Any encountered artifacts were reported by manually delineating the area of the artifacts.These measurements were performed using Fiji Software 68 .The samples analyzed under these conditions were normal saline with 0, 1, 5, and 10% MBs and POL with the same concentrations of MBs.For imaging performed on solidified gels, the TMP containing the samples were incubated 1 h at 37 °C.Entropy, a statistical measure of randomness within the US signal that can be used to characterize the texture of the input image, was also determined utilizing 1 cm 2 rectangular ROIs.Entropy of US images was acquired from MATLAB using e = entropy(I) function were I, is the US image imported into the MATLAB code.Finally, the area of US artifacts was manually measured using Fiji software.
Vascular occlusion and dislodging of gel: Rectangular prism-shaped agar phantoms (4.5 × 11 × 4.5 cm) with an empty cylindrical void (diameter = 0.3 cm; length = 12 cm) to model a vessel were fabricated using a black PVA 3D printed material (Herndon).POL containing 0, 0.1, 1, and 10% MBs was used to occlude the agar-vessel and equilibrated for 5 min at 37 °C.Subsequently, normal saline was passed using a 6 mL syringe with a rate of 1 mL/min while measuring pressure with a sensor (Omega Engineering, Inc, Norwalk, CT, USA).The pressure required to dislodge the gel was recorded.
Ex vivo bovine modeling at physiological temperature: The experiments were performed similarly as previously reported using an ex vivo bovine liver [69][70][71][72] .Briefly, 10 lb bovine livers (Balduccis Market, Bethesda, MD, USA) were submerged for two hours in 10 L of 1 × PBS at 37 C° (Gibco Thermo Fisher Scientific, Waltham, MA, USA).Once the liver reached equilibrium at 37 C°, formulations of POL containing MBs were injected.
Ex vivo bovine liver injections of POL.POL injections were performed using three needle devices: single end hole needle (SEHN, Cook), multiple side hole needle (MSHN, Cook) and multi-pronged injection needles (MPIN, deployed 1 cm, Rex Medical) (Table 3).Needles were guided using a 3D printed needle guide designed to be 45° (Herndon) or freehand, by an experienced radiologist, all with US imaging.The injections were performed with 12 mL syringes (Monoject, Dublin, OH, USA), or 6 mL high pressure syringes (Medallion, Merit Medical, South Jordan, UT, USA) for MPIN needles.Infusions of POL were done with an injection pump (Harvard Apparatus PhD Ultra Syringe Pump, Holliston, MA, USA).
Fig. S17, shows the overall setup needed to perform ex vivo experiments.
From the obtained B-mode US images, measurements of major axis (equatorial distance), minor axis (meridian distance) and area of gels were determined with a customized MATLAB code.Acoustic intensities were acquired by using Fiji software by selecting ROIs manually.Textures such as acoustic heterogeneity and entropy were also obtained from MATLAB software with a customized code by selecting ROIs manually.
Gel dynamics per mL of injected POL: B-mode US images of POL injected, ex vivo, were analyzed using a curvilinear transducer and US equipment (Sonodynamics).POL, 4 mL, containing 0.01% MBs was injected with SEHN (Cook), MSHN (Cook), and MPIN (deployed 1 cm, Rex Medical) at 100 mL/h.The imaging parameters were: gain = 49%, and frequency = 56 Hz.Measurements of the major axis, minor axis, and area were obtained from MATLAB.In addition, eccentricity was calculated with the following formula: Solidities were calculated with MATLAB, and the distance of the centroid of the injection to needle tip was determined manually using Fiji software.
MPIN injection techniques: POL was injected into ex vivo bovine liver with two injection techniques using MPIN (short-tip) (Rex Medical), and MPIN (regular tip) (Rex Medical).Technique one involved utilizing a short tip needle, where the prongs extended 2 cm.As the 4 mL volume was injected, the prongs were gradually pulled back in four steps of 0.5 cm each, retracting from 2 to 0.5 cm without rotating the needle device.Technique two employed a regular tip, advancing the prongs out to a length of 5 cm.Throughout the injection of 4 mL, the prongs were sequentially retracted in 1 cm increments, from 5 cm down to 1 cm, with approximately 0.8 mL administered per step.The device was not rotated during the process.During the injection techniques, the POL distribution was monitored with an EPIQ 7 US system (Philips) using a C5-1 curvilinear transducer and gain of 49%, and frequency of 62 Hz.Gel dynamics and analysis were performed as described above.

Fig. 2 .Fig. 3 .Fig. 4 .
Fig.2.US imaging of 4 mL POL containing MBs injected into bovine liver ex vivo.US image after injection of POL containing 0.01% MBs at 100 mL/hr using (A) SEHN, (B) MSHN, and (C) MPIN -1 cm showing the POL deposition (red arrow) and the needle shaft (white arrow).For the MPIN-1 cm, the based delivery needle is indicated.(D) Acoustic intensities, (E) heterogeneities, (F) entropy, and (G) area of injected POL following injection with different needle devices.(H) MPIN-1 cm injection, 0.1% MBs following POL injection (4 mL, 100 mL/h).The top panel partially shows a deployed needle and the central shaft (white arrows).The margins of the gel are very ill-defined (red arrows).The lower panel shows the gel after removal of the needles.(I) Acoustic intensity, heterogeneity, and entropy of (G).*p < 0.05, ***p < 0.001 from one-way ANOVA statistical test.Error bars represent standard deviations of average (n = 5 for SEHN, n = 4 for MSHN, and n = 3 for MPIN-1cm).

Fig. 5 .
Fig.5.Spatiotemporal distribution of two MPIN injection techniques before and after POL gel injection for POL deposited from the selection of one of the three prongs.(A) US images in B-mode before injection with the MPIN in place with the three needles deployed 2 cm (white arrows points needle tips) and then after completion of the injection of POL while the needles are retracted from 2 to 1 cm tip to tip and removal of the needle (red arrows points gel deposition).(B) Color-coded POL deposition progression per 1 mL increment of the injected volume shown in (A).Green dots of (B) depicts needle tip retraction track.For the injection in (A), the 1 mL incremental values for (C) lengths of major and minor axes, (D) cross-sectional area, and (E) eccentricities are shown.(F) US images in B-mode of MPIN needles before injection (red arrows points gel) and then after needle retraction from 5 to 1 cm while injecting POL gel and needle removal (red arrows points gel deposition).(G) Entropy and acoustic heterogeneity values of (A), and (F) at final 4 mL POL injection.Error bars represent standard deviations for n = 3 average.Dotted lines represent linear regressions.

Fig. 7 .Fig. 8 .
Fig.7.US imageability of POL with DOX, as well as imaging stability over time in ex vivo tissue.(A) B-mode US images of POL with 10 mg/mL of DOX.(B) Area of POL with 10 mg/mL of DOX over time.(C) Acoustic intensity from 0 to 100 min (C).Error bars represent standard deviations for n = 3 average.ns p > 0.999.

Fig. 9 .
Fig.9.US and CT image fusion, and 3D distribution of POL from US and CT imaging.(A) US and CT co-registration from POL with 10 mg/mL (4 mL) injected with MSHN, and their individual US (B), and CT (C) images.(D) 3D distribution of POL gel obtained from US (left), and CT (right) imaging.

5 POLFig. 10 .
Fig.10.Volumetric, morphometric, and surface area-to-volume ratio of POL with and without DOX and comparison between US and CT imaging.Volume of POL with and without 10 mg/mL of DOX obtained from US and CT imaging for (A) SEHN, and (B) MSHN.Sphericity (C), solidity (E), and SA/V (G) are included for SEHN.Sphericity (D), solidity (F), and SA/V (F) are also included for MSHN.*p < 0.05, **p < 0.01, ***p < 0.001 from t-test statistical analysis.

Fig. 11 .
Fig.11.US and CT analysis of 3 mL of POL containing 10 mg/mL in postmortem tissue.(A) B-mode US image of POL injected, as well as its CT (B), and 3D distribution from CT (C).(D) Texture of POL images from US with their area I, major axis (F), minor axis (G), solidity (H), and eccentricity (I) from US and CT imaging.****p < 0.0001 from t-test statistical analysis.Error bars represent standard deviations for n = 3 average.

Table 1 .
Summary of POL characterization containing DOX, iodixanol, and varying MBs*.*Constant concentrations of 10 mg/mL DOX and 40 mg/mL of iodine from iodixanol.

Table 2 .
Volume , sphericity, solidity, and SA/V parameters for POL gel with and without DOX.SA/V = surface area/volume ratio.Values are mean ± standard deviations (n = 3).